Download Super-resolution microscopy of mitochondria

Available online at www.sciencedirect.com
ScienceDirect
Super-resolution microscopy of mitochondria§
Stefan Jakobs1,2 and Christian A Wurm1
Mitochondria, the powerhouses of the cell, are essential
organelles in eukaryotic cells. With their complex inner
architecture featuring a smooth outer and a highly convoluted
inner membrane, they are challenging objects for microscopy.
The diameter of mitochondria is generally close to the
resolution limit of conventional light microscopy, rendering
diffraction-unlimited super-resolution light microscopy
(nanoscopy) for imaging submitochondrial protein distributions
often mandatory. In this review, we discuss what can be
expected when imaging mitochondria with conventional
diffraction-limited and diffraction-unlimited microscopy. We
provide an overview on recent studies using super-resolution
microscopy to investigate mitochondria and discuss further
developments and challenges in mitochondrial biology that
might by addressed with these technologies in the future.
Addresses
1
Department of NanoBiophotonics, Max Planck Institute for Biophysical
Chemistry, 37070 Göttingen, Germany
2
Department of Neurology, University of Göttingen Medical School,
37073 Göttingen, Germany
Corresponding author: Jakobs, Stefan ([email protected])
Current Opinion in Chemical Biology 2014, 20:9–15
This review comes from a themed issue on Molecular imaging
Edited by Christian Eggeling and Mike Heilemann
For a complete overview see the Issue and the Editorial
Available online 25th April 2014
1367-5931/$ – see front matter, # 2014 The Authors. Published by
Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cbpa.2014.03.019
Mitochondria
Mitochondria usually exist within cells as an extended,
dynamic, interconnected network of tubules that is intimately integrated with other cellular compartments [1].
An outer membrane and a highly folded inner membrane
constitute the intricate inner architecture of mitochondria. The invaginations of the inner membrane, called
cristae, are not simply random wide infolds. Rather they
are topologically complicated and their shape and number
is adapted to the cellular requirements. The inner membrane hosts the oxidative phosphorylation system
(OXPHOS). This system facilitates energy conversion
§
This is an open-access article distributed under the terms of the
Creative Commons Attribution-NonCommercial-ShareAlike License,
which permits non-commercial use, distribution, and reproduction in
any medium, provided the original author and source are credited.
www.sciencedirect.com
resulting in the production of ATP, which makes mitochondria indispensable ‘power plants’ of eukaryotic cells.
Since the 1950s, various forms of electron microscopy (EM)
have provided a detailed view on the membrane architecture of these organelles (reviewed, for example, in [2,3]).
EM is exquisitely suited for the investigation of membrane
structures, but is generally less capable of determining the
distribution of individual proteins. Typically, quantitative
immunogold EM requires the decoration of sections with
antibodies, resulting in relatively few gold particles per
decorated section. To determine the suborganellar distribution of a specific protein with this approach, numerous
individual gold localizations are recorded on many images
and an average protein localization is determined [4,5].
Hence immunogold EM is usually not suited to study
protein distribution in individual mitochondria.
Fluorescence microscopy is arguably the most suitable
approach to study the distribution of proteins in single
mitochondria [6]. However, studies using conventional
fluorescence microscopy to investigate protein localizations in these organelles ultimately face the challenge
that mitochondria are small; the width of mitochondrial
tubules is typically between 250 and 500 nm [7–9]. In
conventional (confocal) microscopes diffraction limits
the achievable resolution to 200 nm in the lateral plane
and to 500 nm in the axial direction [10]. Hence the size
of most mitochondria is just at the resolution limit of optical
microscopy making the analysis of submitochondrial
protein distributions always challenging and often entirely
impossible using diffraction limited optical microscopes
[11–15].
Over the last decade several super-resolution microscopy
(nanoscopy) concepts have been devised that allow diffraction-unlimited optical resolution. All concepts that
fundamentally overcome the diffraction limit exploit a
transition between two fluorophore states, usually a fluorescent (on-) and a non-fluorescent (off-) state in order to
discriminate adjacent features. Depending on how the
transition is implemented, the current super-resolution
methods may be assigned to one of two classes, namely
coordinate-targeted (prominent approaches: STED
[16,17], SPEM/SSIM [18,19] and RESOLFT [20–22])
and coordinate-stochastic approaches (PALM [23],
STORM [24], FPALM [25], GSDIM [26], dSTORM
[27], and others). The various methods routinely provide
optical resolution well below 50 nm (i.e. they fundamentally overcome the diffraction barrier), have been implemented with more than one color, and 3D versions are
available. The underlying physical concepts as well as the
Current Opinion in Chemical Biology 2014, 20:9–15
10 Molecular imaging
practical differences between the approaches have been
expertly reviewed elsewhere [28,29,30].
In the three examples, confocal microscopy would fail to
extract any submitochondrial protein distributions. As
expected, isotropic super-resolution would give the
most faithful representation of the starting structure.
However because of their relative complex construction,
microscopes providing true isotropic super-resolution are
currently accessible only in a few specialized labs [32–34].
As shown by the simulations, already an improvement in
the lateral resolution allows detailed additional insight.
Hence currently for many applications 2D super-resolution
microscopy is preferred by many researchers.
The challenge of imaging mitochondria
To evaluate what can be expected when imaging
mitochondria with conventional diffraction-limited
microscopy or diffraction-unlimited nanoscopy, we simulated three simplified models that should reflect differently labeled mitochondria (Figure 1): a mitochondrion
with regularly stacked cristae (crista to crista separation is
100 nm), as often seen in EM images [31] where only
the cristae are labeled (Figure 1b). A helical structure
circumventing the matrix, which might resemble a postulated mitoskeletal element [15] (Figure 1c). Randomly
distributed proteins in the outer membrane (Figure 1d).
We simulated how the labeled mitochondria would
appear in, firstly, a conventional confocal microscope
(i), secondly, a microscope enabling a resolution of
30 nm in the lateral plane and diffraction limited resolution along the optical axis (ii) and finally, a microscope
enabling an isotropic 30 nm resolution (iii). These simulations purposely represent an ideal situation with a bright
signal, no background and no noise. Hence, in reality, the
obtainable images may look worse, but not better.
Super-resolution microscopy of mitochondria
A number of studies using light microscopy with
increased, albeit not diffraction unlimited resolution,
demonstrated the advantages of improved resolution
when imaging mitochondria. 4Pi-microscopy, which
increases the resolution along the z-axis to 100 nm,
allowed better representations of the overall structure
of the mitochondrial network both in living yeast cells
[9,35], as well as in chemically fixed human cells lines
[36–38]. Likewise, 2D and 3D structured illumination,
which can improve the resolution by a factor of about two,
has been used to better represent mitochondrial networks
Figure 1
(a)
(b)
(c)
(d)
Z
X
PSF
Y
i
Y
Z
conf.
Z
X
X
Y
Z
X
X
Y
X
X
ii
Z
2D SR
Z
Y
X
X
Y
X
Z
X
Y
X
X
iii
Z
3D SR
Y
X
Z
X
Y
X
Z
X
Y
X
X
Current Opinion in Chemical Biology
Simulations. (a) Point spread functions (PSFs) drawn to scale of (i) a confocal microscope (diffraction limited), (ii) a super-resolution (SR) microscope
enabling a resolution of 30 nm in the optical plane and conventional resolution along the z-axis, and (iii) a super-resolution microscope enabling
isotropic resolution of 30 nm. This resolution was chosen because it can be attained with various instruments and because antibody labeling using a
primary and a dye labeled secondary antibody would increase any structure to about this size [69,70]. (b–d) Simulations on how different labeled
mitochondrial structures would appear in the microscopic image. To this end, the simulated data (top: 3D rendering of the data) were convolved with
the respective PSFs. Shown are xy and xz sections. The simulated mitochondria have a diameter of 220 nm (b,c; inner membrane) and 280 nm (d;
outer membrane). Scale bars: 500 nm.
Current Opinion in Chemical Biology 2014, 20:9–15
www.sciencedirect.com
Super-resolution microscopy of mitochondria Jakobs and Wurm 11
in living cells [39,40]. Although these methods improve
the resolution compared to confocal microscopy, they do
not allow substantially better resolution than 100 nm.
These methods have thus not established as routine tools
to study submitochondrial protein distributions.
Cells with labeled mitochondria have been used in several early implementations of super-resolution microscopy, including the first manuscript using PALM
microscopy [23] and the first manuscript demonstrating
two-color STED microscopy [41]. isoSTED microscopy
enabling isotropic 3D resolution of 30–40 nm was used to
reveal the distributions of several proteins within the
organelle and allowed the visualization of individual
cristae [32,42]. Utilizing STORM, Shim et al. succeeded
in visualizing mitochondrial inner membrane dynamics in
living cells using MitoTracker Red, a photoswitchable
membrane probe [43].
Outer membrane proteins
Tom20 is a subunit of the translocase of the mitochondrial
outer membrane (TOM) complex, which is the major
import gate for nuclear encoded proteins into mitochondria. Several studies have been using antisera against
Tom20 to highlight the outer membrane or to study the
distribution of the TOM complex itself [32,41,44,45].
Conclusively, these studies showed clustered distribution
of the TOM complexes, which is concealed in diffractionlimited imaging, essentially as can be seen in the simulated
image data (Figure 1d).
Huang and colleagues imaged cells immunolabeled for
Tom20 and beta-tubulin by multicolor 3D STORM and
provided detailed view of the intricate morphology of the
entire mitochondrial network in chemically fixed monkey
cells [45]. This study provided detailed insights into the
nanoscale spatial arrangement between mitochondria and
the microtubule cytoskeleton. Interestingly, some mitochondria that appeared to co-align with microtubules
when imaged with conventional microscopy were shown
to have distinct interaction sites which were spaced by
stretches of noncontact regions (Figure 2a).
In a high-throughput STED study involving more than
1000 cells we demonstrated that the clustering of the
TOM complexes in the outer membrane is adjusted to
the cellular growth conditions [44]. Differences in the
density of the clusters in the outer membrane were
observed in cell lines having different growth rates. Likewise, a difference was recorded for cells forming a small
colony of 20–30 cells: The clusters were sparser in the
cells in the center of the colony than at its rim. Somewhat
unexpectedly, this study also revealed that the density of
TOM clusters followed an inner-cellular gradient from
Figure 2
(a)
STORM
(b)
STED
(c)
GSDIM
(d)
STED
Current Opinion in Chemical Biology
Super-resolution microscopy of protein distributions in mammalian mitochondria. (a) Two-color STORM images showing the interaction between
mitochondria (magenta) and microtubules (green) [45]. (b) Two-color STED microscopy of hVDAC3 and hexokinase-I [47]. (c,d) GSDIM and STED
imaging of Mic60 (mitofilin) showing the peculiar ordered arrangement of the MICOS clusters [31]. Scale bars: 1000 nm.
www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 20:9–15
12 Molecular imaging
the perinuclear to the peripheral mitochondria.
Altogether, the reported findings showed a correlation
of the metabolic activity of the cells and the nanoscale
clustering of TOM. This suggests that the control of the
distribution of TOM might be a mechanism to regulate
protein import into mitochondria.
The voltage-dependent anion channel (VDAC, also
known as mitochondrial porin) is the major transport
channel mediating the transport of metabolites, including
ATP, across the outer membrane [46]. In humans, three
isoforms (hVDAC1, hVDAC2, hVDAC3) exist which are
suggested to bind the cytosolic protein hexokinase-I.
Dual-color STED microscopy of immunolabelled
U2OS cells showed that the extent of colocalization
between the hexokinase-I and hVDAC is isoform-specific
(Figure 2b). This observation suggests functional differences between the three VDAC isoforms [47].
Inner membrane proteins
The inner membrane exhibits two structural domains, the
inner boundary membrane that is parallel to the outer
membrane and the cristae membrane. Only recently it
was nonambiguously demonstrated that the cristae membrane and the inner boundary membrane have different
protein compositions [4,5,48–50].
Few studies have investigated the nanoscale distribution
of proteins in the mitochondrial inner membrane with
light microscopy [23,32,51,52] and mainly concentrated
on proteins in OXPHOS, presumably because of the
abundance and the relative ease of labeling of these
proteins. One exception is a study using EM and STED
microscopy to investigate the submitochondrial distribution of a protein complex named MICOS (mitochondrial contact site and cristae organizing system; previously
also called MINOS or MitOS) [31]. MICOS is a large
hetero-oligomeric protein complex that has crucial roles
in the maintenance of cristae junctions, inner membrane
architecture and in the formation of contact sites to the
outer membrane [53]. STED as well as GSDIM imaging
of primary human fibroblasts decorated with antibodies
against the MICOS core component Mic60 (according to
a recently unified nomenclature [54]; previous name:
mitofilin) showed that MICOS forms distinct clusters
(Figure 2c,d). Unexpectedly, these clusters were arranged
in a regularly spaced array in parallel to the cell growth
surface. By quantitative immunogold EM we demonstrated that Mic60 is preferentially enriched at the cristae
junctions [31]. Furthermore, electron tomography
showed a horizontal arrangement of cristae junctions in
many mitochondria. Altogether this demonstrates that at
least in the peripheral mitochondria of human fibroblasts
the inner-mitochondrial localization of MICOS is correlated to the orientation of the cellular growth surface,
suggesting an unexpected level of regulation of inner
mitochondrial architecture.
Current Opinion in Chemical Biology 2014, 20:9–15
Nucleoids
Mitochondria contain their own genome (mtDNA), which
is packaged into nucleoprotein complexes (nucleoids)
located in the innermost mitochondrial compartment,
the aqueous matrix [55,56]. The nucleoids are distributed
throughout the mitochondrial network. In humans, the
mtDNA encodes 13 proteins, which are essential for the
function of OXPHOS. An important and still not conclusively answered question is whether a single nucleoid
contains one or multiple mtDNA copies [57].
This issue has been addressed by a number of studies
using different experimental procedures, which came to
rather inconclusive estimates, ranging from on average
1.4–10 mtDNA molecules per individual nucleoid
(reviewed in [55,56]). STED microscopy allowed to
visualize 1.6 times more nucleoids per human fibroblast
than confocal microscopy (Figure 3a,b). Using automated
image analysis we found 1883 106 nucleoids per
primary human fibroblast [58]. Based on this data combined with the average number of mtDNAs per cell as
determined by molecular biology, the average number of
mtDNA molecules per nucleoid was calculated as 1.4.
This number is smaller than most other estimates, which
may be due to the fact that super-resolution microscopy
allowed to count the number of nucleoids more precisely.
In addition, there might be differences in the number of
mtDNAs per nucleoid in different cell lines or tissues.
Hence more studies are required to allow a definite
answer on the average number of mtDNAs per nucleoid.
In a careful study employing 2D and 3D photoactivated
localization microscopy (PALM and iPALM, respectively),
Brown et al. [59] demonstrated that nucleoids often adopt
an ellipsoidal shape (Figure 3c,d), although their shape
may vary strongly and may depend on the interaction of the
nucleoid with the inner membrane. They determined the
mean dimensions of an ellipsoidal nucleoid as
85 nm 108 nm 146 nm. From these data they concluded that the mtDNA is extraordinarily condensed,
similar to the situation in a papillomavirus capsid [56,59].
In an independent STED study, Kukat et al. semi-automatically analyzed the size of >35 000 nucleoids in seven
different cells lines [58]. Fully in line with the study by
Brown et al., this study also demonstrated a large variability
in the shape and size of the nucleoids. Assuming the
simplified model of a spherical nucleoid, it was determined
that the antibody decorated nucleoids had a diameter of
100 nm, also in good agreement with the study be Brown
et al. Interestingly, the mean diameter was well conserved
across several cultured mammalian cell lines.
In a technical tour de force, Kopek et al. correlated 3D
super-resolution (iPALM) images of mitochondrial
nucleoids with 3D EM data [60]. Using a modified
Tokuyasu cryosectioning protocol for fixation and freezing,
www.sciencedirect.com
Super-resolution microscopy of mitochondria Jakobs and Wurm 13
Figure 3
(a)
confocal
iPALM
(c)
Y
(b)
iPALM
(d)
STED
PALM & EM
(e)
Z
X
X
Current Opinion in Chemical Biology
Super-resolution microscopy of mitochondrial nucleoids. (a,b) Nucleoids were labeled with antibodies against dsDNA and imaged with a confocal or a
STED microscope [58]. (c,d) Ellipsoidal nucleoid projections recorded with iPALM [59]. (e) Correlated image of TFAM-mEos2 PALM data with electron
micrograph [63]. Scale bars: 1000 nm (a,b), 150 nm (c), and 200 nm (d,e).
they prepared 500–750 nm thick slices of cells expressing
TFAM fused to the photoconvertible protein mEOS2.
These slices were imaged with iPALM [33] to record
the 3D distribution of TFAM in the slice. Next, 3D
EM images were obtained with focused ion beam blockface ablation followed by scanning EM imaging. Using this
approach Kopek et al. observed a variety of nucleoid sizes
and shapes. In some cases cristae and nucleoids appeared to
be intertwined in a complex manner. Understanding the
biological relevance of these observations would require a
lot more image recordings, which presumably would be a
considerable challenge given the technical complexity of
the chosen approach. However, technically less demanding 2D approaches correlating various super-resolution
microscopy approaches with EM have been developed
by the same group and others (Figure 3e), opening up this
technology to a wider community [23,61–63].
analyses to evaluate the heterogeneity on the nanoscale
[44,64,65], in analyzing protein movements [52,66], as
well as in counting the number of molecules [67,68]. Each
of these tasks represents a formidable challenge, but the
first important steps have been taken and given the
impressive progress that super-resolution has made over
the last decade, the challenges seem surmountable.
Figure 4
Mitochondrial architecture
How are cristae established, shaped and maintained?
Do mitochondria have structural elements corresponding to a
skeleton?
Heterogeneity of mitochondria
Are all mitochondria / nucleoids alike in a single cell?
What is the level of heterogeneity between cells?
Mitochondrial inner membrane
Conclusions and perspectives
Given the tremendous benefits that super-resolution offers
for imaging mitochondria, we are undoubtedly going to see
many more studies using these technologies to tackle
fundamental problems in mitochondrial biology in the
near future. There are many issues where super-resolution
is needed; some of those that we feel are amongst the most
current ones are highlighted in Figure 4.
How is the heterogeneity of the inner membrane established
and maintained? How do proteins move within the various
mitochondrial compartments? Do cristae junctions act as
barriers?
Assembly of OXPHOS supercomplexes
Where are the nuclear and mitochondrial encoded subunits of
OXPHOS supercomplexes inserted? Where and when is
assembly of the supercomplexes taking place?
Current Opinion in Chemical Biology
Answering these questions will require further progress in
(semi-)automated super-resolution microscopy and image
www.sciencedirect.com
Future challenges.
Current Opinion in Chemical Biology 2014, 20:9–15
14 Molecular imaging
Acknowledgements
We thank J Jethwa for critical reading of the manuscript. SJ acknowledges
support by the Cluster of Excellence and DFG Research Center Nanoscale
Microscopy and Molecular Physiology of the Brain.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
of special interest
of outstanding interest
1.
Nunnari J, Suomalainen A: Mitochondria: in sickness and in
health. Cell 2012, 148:1145-1159.
2.
Frey TG, Mannella CA: The internal structure of mitochondria.
Trends Biochem Sci 2000, 25:319-324.
3.
Scheffler IE: Mitochondria. edn 2. Hoboken, NJ, USA: John Wiley
& Sons, Inc.; 2008, .
4.
Vogel F, Bornhovd C, Neupert W, Reichert AS: Dynamic
subcompartmentalization of the mitochondrial inner
membrane. J Cell Biol 2006, 175:237-247.
5.
Stoldt S, Wenzel D, Hildenbeutel M, Wurm CA, Herrmann JM,
Jakobs S: The inner-mitochondrial distribution of Oxa1
depends on the growth conditions and on the availability of
substrates. Mol Biol Cell 2012, 23:2292-2301.
6.
Jakobs S: High resolution imaging of live mitochondria.
Biochim Biophys Acta 2006, 1763:561-575.
7.
Kaasik A, Safiulina D, Zharkovsky A, Veksler V: Regulation of
mitochondrial matrix volume. Am J Physiol Cell Physiol 2007,
292:C157-C163.
8.
Jakobs S, Stoldt S, Neumann D: Light microscopic analysis
of mitochondrial heterogeneity in cell populations and
within single cells. Adv Biochem Eng Biotechnol 2011, 124:1-19.
9.
Egner A, Jakobs S, Hell SW: Fast 100-nm resolution threedimensional microscope reveals structural plasticity of
mitochondria in live yeast. Proc Natl Acad Sci U S A 2002,
99:3370-3375.
10. Born M, Wolf E: Principles of Optics. edn 7. Cambridge, UK:
Cambridge University Press; 2002, .
11. Dikov D, Bereiter-Hahn J: Inner membrane dynamics in
mitochondria. J Struct Biol 2013, 183:455-466.
12. Muster B, Kohl W, Wittig I, Strecker V, Joos F, Haase W, BereiterHahn J, Busch K: Respiratory chain complexes in dynamic
mitochondria display a patchy distribution in life cells. PLoS
ONE 2010, 5:e11910.
13. Jimenez L, Laporte D, Duvezin-Caubet S, Courtout F, Sagot I:
Mitochondrial ATP synthases cluster as discrete domains that
reorganize with the cellular demand for oxidative
phosphorylation. J Cell Sci 2013.
14. Schauss AC, Bewersdorf J, Jakobs S: Fis1p and Caf4p, but not
Mdv1p, determine the polar localization of Dnm1p clusters on
the mitochondrial surface. J Cell Sci 2006, 119:3098-3106.
15. Hoppins S, Collins SR, Cassidy-Stone A, Hummel E, Devay RM,
Lackner LL, Westermann B, Schuldiner M, Weissman JS,
Nunnari J: A mitochondrial-focused genetic interaction map
reveals a scaffold-like complex required for inner membrane
organization in mitochondria. J Cell Biol 2011, 195:323-340.
16. Hell SW, Wichmann J: Breaking the diffraction resolution limit
by stimulated emission: stimulated-emission-depletion
fluorescence microscopy. Opt Lett 1994, 19:780-782.
17. Klar TA, Jakobs S, Dyba M, Egner A, Hell SW: Fluorescence
microscopy with diffraction resolution barrier broken by
stimulated emission. Proc Natl Acad Sci U S A 2000, 97:82068210.
18. Gustafsson MG: Nonlinear structured-illumination microscopy:
wide-field fluorescence imaging with theoretically unlimited
resolution. Proc Natl Acad Sci U S A 2005, 102:13081-13086.
Current Opinion in Chemical Biology 2014, 20:9–15
19. Heintzmann R, Jovin TM, Cremer C: Saturated patterned excitation
microscopy — a concept for optical resolution improvement. J
Opt Soc Am A Opt Image Sci Vis 2002, 19:1599-1609.
20. Hell SW: Toward fluorescence nanoscopy. Nat Biotechnol 2003,
21:1347-1355.
21. Brakemann T, Stiel AC, Weber G, Andresen M, Testa I,
Grotjohann T, Leutenegger M, Plessmann U, Urlaub H, Eggeling C
et al.: A reversibly photoswitchable GFP-like protein with
fluorescence excitation decoupled from switching. Nat
Biotechnol 2011, 29:942-947.
22. Grotjohann T, Testa I, Leutenegger M, Bock H, Urban NT, LavoieCardinal F, Willig KI, Eggeling C, Jakobs S, Hell SW: Diffractionunlimited all-optical imaging and writing with a photochromic
GFP. Nature 2011, 478:204-208.
23. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S,
Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF:
Imaging intracellular fluorescent proteins at nanometer
resolution. Science 2006, 313:1642-1645.
24. Rust M, Bates M, Zhuang X: Sub-diffraction-limit imaging by
stochastic optical reconstruction microscopy (STORM). Nat
Methods 2006, 3:793-795.
25. Hess ST, Girirajan TP, Mason MD: Ultra-high resolution imaging
by fluorescence photoactivation localization microscopy.
Biophys J 2006, 91:4258-4272.
26. Fölling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S,
Eggeling C, Hell SW: Fluorescence nanoscopy by ground-state
depletion and single-molecule return. Nat Methods 2008,
5:943-945.
27. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B,
Mukherjee A, Tinnefeld P, Sauer M: Subdiffraction-resolution
fluorescence imaging with conventional fluorescent probes.
Angew Chem Int Ed Engl 2008, 47:6172-6176.
28. Hell SW: Far-field optical nanoscopy. Single Molecule
Spectroscopy in Chemistry, Physics and Biology. Berlin, Germany:
Springer; 2009, 298-365.
This review explains that all super-resolution concepts that fundamentally
overcome the diffraction barrier exploit a transition between two fluorophore states (generally a bright and a dark state) to discriminate adjacent
molecules.
29. Huang B, Bates M, Zhuang X: Super-resolution fluorescence
microscopy. Annu Rev Biochem 2009, 78:993-1016.
A comprehensive well-written review describing all current super-resolution approaches.
30. Toomre D, Bewersdorf J: A new wave of cellular imaging. Annu
Rev Cell Dev Biol 2010, 26:285-314.
31. Jans DC, Wurm CA, Riedel D, Wenzel D, Stagge F, Deckers M,
Rehling P, Jakobs S: STED super-resolution microscopy
reveals an array of MINOS clusters along human
mitochondria. Proc Natl Acad Sci U S A 2013, 110:8936-8941.
STED super-resolution microscopy and electron microscopy were used
to determine the submitochondrial localization of the MICOS (MINOS,
MitOS) complex in mitochondria of primary fibroblasts. The study
revealed a regularly spaced pattern of MICOS clusters arranged in parallel
to the cell growth surfaces in peripheral mitochondria.
32. Schmidt R, Wurm CA, Jakobs S, Engelhardt J, Egner A, Hell SW:
Spherical nanosized focal spot unravels the interior of cells.
Nat Methods 2008, 5:539-544.
33. Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J,
Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P,
Davidson MW et al.: Interferometric fluorescent super-resolution
microscopy resolves 3D cellular ultrastructure. Proc Natl Acad
Sci U S A 2009, 106:3125-3130.
34. Aquino D, Schonle A, Geisler C, Middendorff CV, Wurm CA,
Okamura Y, Lang T, Hell SW, Egner A: Two-color nanoscopy of
three-dimensional volumes by 4Pi detection of stochastically
switched fluorophores. Nat Methods 2011, 8:353-359.
35. Gugel H, Bewersdorf J, Jakobs S, Engelhardt J, Storz R, Hell SW:
Cooperative 4Pi excitation and detection yields sevenfold
sharper optical sections in live-cell microscopy. Biophys J
2004, 87:4146-4152.
www.sciencedirect.com
Super-resolution microscopy of mitochondria Jakobs and Wurm 15
36. Medda R, Jakobs S, Hell SW, Bewersdorf J: 4Pi microscopy of
quantum dot-labeled cellular structures. J Struct Biol 2006,
156:517-523.
53. van der Laan M, Bohnert M, Wiedemann N, Pfanner N: Role of
MINOS in mitochondrial membrane architecture and
biogenesis. Trends Cell Biol 2012, 22:185-192.
37. Dlaskova A, Spacek T, Santorova J, Plecita-Hlavata L, Berkova Z,
Saudek F, Lessard M, Bewersdorf J, Jezek P: 4Pi microscopy
reveals an impaired three-dimensional mitochondrial network
of pancreatic islet beta-cells, an experimental model of type-2
diabetes. Biochim Biophys Acta 2010, 1797:1327-1341.
54. Pfanner N, van der Laan M, Amati P, Capaldi RA, Caudy AA,
Chacinska A, Darshi M, Deckers M, Hoppins S, Icho T et al.:
Uniform nomenclature for the mitochondrial contact site and
cristae organizing system. J Cell Biol 2014, 204:1083-1086.
38. Plecita-Hlavata L, Lessard M, Santorova J, Bewersdorf J, Jezek P:
Mitochondrial oxidative phosphorylation and energetic status
are reflected by morphology of mitochondrial network in INS1E and HEP-G2 cells viewed by 4Pi microscopy. Biochim
Biophys Acta 2008, 1777:834-846.
39. Hirvonen LM, Wicker K, Mandula O, Heintzmann R: Structured
illumination microscopy of a living cell. Eur Biophys J 2009,
38:807-812.
40. Shao L, Kner P, Rego EH, Gustafsson MG: Super-resolution 3D
microscopy of live whole cells using structured illumination.
Nat Methods 2011, 8:1044-1046.
41. Donnert G, Keller J, Wurm CA, Rizzoli SO, Westphal V, Schonle A,
Jahn R, Jakobs S, Eggeling C, Hell SW: Two-color far-field
fluorescence nanoscopy. Biophys J 2007, 92:L67-L69.
42. Schmidt R, Wurm CA, Punge A, Egner A, Jakobs S, Hell SW:
Mitochondrial cristae revealed with focused light. Nano Lett
2009, 9:2508-2510.
43. Shim SH, Xia C, Zhong G, Babcock HP, Vaughan JC, Bo Huang B,
Wang X, Xu C, Bi GQ, Zhuang X: Super-resolution fluorescence
imaging of organelles in live cells with photoswitchable
membrane probes. Proc Natl Acad Sci U S A 2012, 109:13978-13983.
44. Wurm CA, Neumann D, Lauterbach MA, Harke B, Egner A,
Hell SW, Jakobs S: Nanoscale distribution of mitochondrial
import receptor Tom20 is adjusted to cellular conditions and
exhibits an inner-cellular gradient. Proc Natl Acad Sci U S A
2011, 108:13546-13551.
One of the first studies using quantitative super-resolution microscopy
demonstrating an inner-cellular gradient in the nanoscale distribution of
the mitochondrial TOM complex.
45. Huang B, Jones SA, Brandenburg B, Zhuang X: Whole-cell 3D
STORM reveals interactions between cellular structures
with nanometer-scale resolution. Nat Methods 2008, 5:1047-1052.
The manuscript describes the use of multicolor 3D STORM microscopy to
record whole-cell images with a spatial resolution of 20–30 nm and 60–
70 nm in the lateral and axial dimensions, respectively. The relationship
between mitochondria and microtubules is shown.
46. Colombini M: VDAC structure, selectivity, and dynamics.
Biochim Biophys Acta 2012, 1818:1457-1465.
47. Neumann D, Buckers J, Kastrup L, Hell SW, Jakobs S: Two-color
STED microscopy reveals different degrees of colocalization
between hexokinase-I and the three human VDAC isoforms.
PMC Biophys 2010, 3:4.
48. Gilkerson RW, Selker JML, Capaldi RA: The cristal membrane of
mitochondria is the principal site of oxidative phosphorylation.
FEBS Lett 2003, 546:355-358.
49. Wurm CA, Jakobs S: Differential protein distributions define
two subcompartments of the mitochondrial inner membrane
in yeast. FEBS Lett 2006, 580:5628-5634.
50. Suppanz IE, Wurm CA, Wenzel D, Jakobs S: The m-AAA protease
processes cytochrome c peroxidase preferentially at the inner
boundary membrane of mitochondria. Mol Biol Cell 2009,
20:572-580.
51. van de Linde S, Sauer M, Heilemann M: Subdiffractionresolution fluorescence imaging of proteins in the
mitochondrial inner membrane with photoswitchable
fluorophores. J Struct Biol 2008, 164:250-254.
52. Appelhans T, Richter CP, Wilkens V, Hess ST, Piehler J, Busch KB:
Nanoscale organization of mitochondrial microcompartments
revealed by combining tracking and localization microscopy.
Nano Lett 2012, 12:610-616.
Tracking of single molecules revealed diffusion-restricting microcompartments in the inner membrane of mitochondria in living cells.
www.sciencedirect.com
55. Kukat C, Larsson NG: mtDNA makes a U-turn for the
mitochondrial nucleoid. Trends Cell Biol 2013, 23:457-463.
56. Bogenhagen DF: Mitochondrial DNA nucleoid structure.
Biochim Biophys Acta 2012, 1819:914-920.
57. Friedman JR, Nunnari J: Mitochondrial form and function.
Nature 2014, 505:335-343.
58. Kukat C, Wurm CA, Spahr H, Falkenberg M, Larsson NG,
Jakobs S: Super-resolution microscopy reveals that
mammalian mitochondrial nucleoids have a uniform size and
frequently contain a single copy of mtDNA. Proc Natl Acad Sci U
S A 2011, 108:13534-13539.
59. Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF,
Hess HF, Clayton DA: Superresolution fluorescence imaging of
mitochondrial nucleoids reveals their spatial range, limits, and
membrane interaction. Mol Cell Biol 2011, 31:4994-5010.
60. Kopek BG, Shtengel G, Xu CS, Clayton DA, Hess HF: Correlative
3D superresolution fluorescence and electron microscopy
reveal the relationship of mitochondrial nucleoids to
membranes. Proc Natl Acad Sci U S A 2012, 109:6136-6141.
This manuscript reports on the correlation of 3D localization microscopy
with scanning EM images generated by focused ion beam ablation. In
provides unique insight into the relationship of nucleoids to the inner
membrane.
61. Watanabe S, Punge A, Hollopeter G, Willig KI, Hobson RJ,
Davis MW, Hell SW, Jorgensen EM: Protein localization in
electron micrographs using fluorescence nanoscopy. Nat
Methods 2011, 8:80-84.
62. Nanguneri S, Flottmann B, Horstmann H, Heilemann M, Kuner T:
Three-dimensional, tomographic super-resolution
fluorescence imaging of serially sectioned thick samples.
PLoS ONE 2012, 7:e38098.
63. Kopek BG, Shtengel G, Grimm JB, Clayton DA, Hess HF:
Correlative photoactivated localization and scanning electron
microscopy. PLoS ONE 2013, 8:e77209.
64. Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL,
Lippincott-Schwartz J: Probing protein heterogeneity in the
plasma membrane using PALM and pair correlation analysis.
Nat Methods 2011, 8:969-975.
65. Owen DM, Williamson DJ, Boelen L, Magenau A, Rossy J, Gaus K:
Quantitative analysis of three-dimensional fluorescence
localization microscopy data. Biophys J 2013, 105:L05-L07.
665. Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E,
Lippincott-Schwartz J: High-density mapping of singlemolecule trajectories with photoactivated localization
microscopy. Nat Methods 2008, 5:155-157.
67. Puchner EM, Walter JM, Kasper R, Huang B, Lim WA: Counting
molecules in single organelles with superresolution
microscopy allows tracking of the endosome maturation
trajectory. Proc Natl Acad Sci U S A 2013, 110:16015-16020.
68. Lee SH, Shin JY, Lee A, Bustamante C: Counting single
photoactivatable fluorescent molecules by photoactivated
localization microscopy (PALM). Proc Natl Acad Sci U S A 2012,
109:17436-17441.
69. Weber K, Rathke PC, Osborn M: Cytoplasmic microtubular
images in glutaraldehyde-fixed tissue culture cells by electron
microscopy and by immunofluoresence microscopy. Proc Natl
Acad Sci U S A 1978, 75:1820-1824.
70. Dyba M, Jakobs S, Hell SW: Immunofluorescence stimulated
emission depletion microscopy. Nat Biotechnol 2003,
21:1303-1304.
Current Opinion in Chemical Biology 2014, 20:9–15